CD4 T-Cell Help Programs a Change in CD8 T-Cell Function Enabling Effective Long-Term Control of Murine Gammaherpesvirus 68: Role of PD-1-PD-L1 Interactions

We previously showed that agonistic antibodies to CD40 could substitute for CD4 T-cell help and prevent reactivation of murine gammaherpesvirus 68 (MHV-68) in the lungs of major histocompatibility complex (MHC) class II^−/− (CII^−/−) mice, which are CD4 T cell deficient. Although CD8 T cells were required for this effect, no change in their activity was detected in vitro. A key question was whether anti-CD40 treatment (or CD4 T-cell help) changed the function of CD8 T cells or another cell type in vivo. To address this question, in the present study, we showed that adoptive transfer of CD8 T cells from virus-infected wild-type mice or anti-CD40-treated CII^−/− mice caused a significant reduction in lung viral titers, in contrast to those from control CII^−/− mice. Anti-CD40 treatment also greatly prolonged survival of infected CII^−/− mice. This confirms that costimulatory signals cause a change in CD8 T cells enabling them to maintain effective long-term control of MHV-68. We investigated the nature of this change and found that expression of the inhibitory receptor PD-1 was significantly increased on CD8 T cells in the lungs of MHV-68-infected CII^−/−, CD40^−/−, or CD80/86^−/− mice, compared with that in wild-type or CD28/CTLA4^−/− mice, correlating with the level of viral reactivation. Furthermore, blocking PD-1-PD-L1 interactions significantly reduced viral reactivation in CD4 T-cell-deficient mice. In contrast, the absence of another inhibitory receptor, NKG2A, had no effect. These data suggest that CD4 T-cell help programs a change in CD8 T-cell function mediated by altered PD-1 expression, which enables effective long-term control of MHV-68.Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen which is closely related to Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV) (17, 64). Intranasal administration of MHV-68 to mice results in acute productive infection of lung epithelial cells and a latent infection in various cell types, including B lymphocytes, dendritic cells, epithelial cells, and macrophages (18, 19, 52, 53, 61, 65). The virus induces an inflammatory infiltrate in the lungs, lymph node enlargement, splenomegaly, and mononucleosis comprising increased numbers of activated CD8 T cells in the blood (53, 58). It has also been reported to induce lymphoproliferative disease/lymphoma in immunocompromised mice (30, 55, 60). Thus, the pathogenesis resembles that of EBV in humans, although structurally, the virus is more closely related to KSHV.Infectious MHV-68 is cleared from the lungs by a T-cell-dependent mechanism 10 to 15 days after infection (18, 53, 56). In wild-type mice, the lungs remain clear of replicating virus thereafter. Although CD4 T cells are not essential for primary clearance of replicating virus, they are required for effective long-term control (11). Thus, major histocompatibility complex (MHC) class II^−/− mice that lack CD4 T cells or mice rendered CD4 deficient by antibody treatment initially clear infectious virus from the lungs. However, infectious virus reactivates in the lungs 10 to 15 days later and gradually increases in titer (11, 43). The infected CD4-deficient mice eventually die, apparently from long-term lung damage due to continuing lytic viral replication (11). MHC class II^−/− mice do not produce antibody to T-dependent antigens (10). Cytotoxic T-lymphocyte (CTL) epitopes have been identified in open reading frame (ORF) 6 (p56, H-2D^b-restricted), and ORF 61 (p79, H-2K^b-restricted) gene products, which appear to encode early lytic-phase proteins (32, 49). The epitopes are presented during two distinct phases during MHV-68 infection, which changes the pattern of CTL dominance (32, 51). However, there is no significant difference in the numbers of CD8 T cells specific for each epitope in wild-type mice and CD4 T-cell-deficient mice (4, 50). In addition, CTL activity measured in vitro does not differ substantially in the lungs of wild-type mice or CD4 T-cell-deficient mice (4, 11, 50). Furthermore, postexposure vaccination with the p56 epitope failed to prevent viral reactivation in class II^−/− mice, despite dramatically expanding the number of CD8 T cells specific for the peptide (5). In contrast, vaccination of wild-type mice against these epitopes reduced lytic viral titers in the lung dramatically on subsequent challenge with MHV-68. B-cell-deficient mice clear MHV-68 with the kinetics of wild-type mice and do not show viral reactivation in the lungs (13, 61), suggesting that antibody is not essential for control of the virus. Depletion of CD4 T cells during the latent phase of infection in B-cell-deficient mice does not induce viral reactivation, whereas depletion of both CD4 and CD8 T-cell subsets provokes viral reactivation in the lungs (52). Short-term depletion of both CD4 and CD8 T-cell subsets during the latent phase of infection in wild-type mice does not lead to viral reactivation probably due to the presence of neutralizing antibody (11). Taken together, these results suggest that CD4 and CD8 T cells and B cells play overlapping roles in preventing or controlling reactivation of MHV-68 during the latent phase of infection. However, the B-cell- and CD8 T-cell-mediated control mechanisms do not develop in the absence of CD4 T cells.We, and others, have previously shown that the costimulatory molecule CD28 is not required for long-term control of MHV-68 (28, 29). However, interestingly, mice lacking both of the ligands for CD28, CD80 and CD86, show viral reactivation in the lung (21, 35). Our previously published data showed that agonistic antibodies to CD40 could substitute for CD4 T-cell function in the long-term control of MHV-68 (46). CD8 T-cell receptor-positive (TCR+) cells were required for this effect, while antibody production was not restored (45, 46). MHV-68-infected CD40L^−/− mice (7) and CD40^−/− mice (29) also showed viral reactivation in the lungs. However, no change in CD8 CTL activity was detected in in vitro assays following anti-CD40 treatment (46). A key question was whether anti-CD40 treatment (or CD4 T-cell help) caused a direct change in CD8 T-cell function or whether both CD8 T cells and an independent anti-CD40-sensitive step were required for viral control. To address this question, we used adoptive transfer of CD8 T cells from MHV-68-infected wild-type mice, anti-CD40-treated mice, or control MHC class II^−/− mice to MHV-68-infected class II^−/− recipients. We also investigated whether anti-CD40 treatment prolonged survival in addition to reducing lung viral titers. The heterodimeric molecule CD94/NKG2A has been implicated in negatively regulating the CD8 T-cell response to polyomavirus (38) and herpes simplex virus (HSV) (54), while the inhibitory receptor PD-1 (programmed death 1) has been implicated in T-cell exhaustion following infection with several other persistent viruses (2, 15, 20, 22, 26, 36, 39-41, 57, 67). In the present study, we investigated the effect of signaling via various costimulatory molecules on the expression of NKG2A and PD-1 and how these molecules influenced viral control.     

Novel Denitrifying Bacterium Ochrobactrum anthropi YD50.2 Tolerates High Levels of Reactive Nitrogen Oxides

Most studies of bacterial denitrification have used nitrate (NO₃⁻) as the first electron acceptor, whereas relatively less is understood about nitrite (NO₂⁻) denitrification. We isolated novel bacteria that proliferated in the presence of high levels of NO₂⁻ (72 mM). Strain YD50.2, among several isolates, was taxonomically positioned within the α subclass of Proteobacteria and identified as Ochrobactrum anthropi YD50.2. This strain denitrified NO₂⁻, as well as NO₃⁻. The gene clusters for denitrification (nar, nir, nor, and nos) were cloned from O. anthropi YD50.2, in which the nir and nor operons were linked. We confirmed that nirK in the nir-nor operon produced a functional NO₂⁻ reductase containing copper that was involved in bacterial NO₂⁻ reduction. The strain denitrified up to 40 mM NO₂⁻ to dinitrogen under anaerobic conditions in which other denitrifiers or NO₃⁻ reducers such as Pseudomonas aeruginosa and Ralstonia eutropha and nitrate-respiring Escherichia coli neither proliferated nor reduced NO₂⁻. Under nondenitrifying aerobic conditions, O. anthropi YD50.2 and its type strain ATCC 49188^T proliferated even in the presence of higher levels of NO₂⁻ (100 mM), and both were considerably more resistant to acidic NO₂⁻ than were the other strains noted above. These results indicated that O. anthropi YD50.2 is a novel denitrifier that has evolved reactive nitrogen oxide tolerance mechanisms.Environmental bacteria maintain the global nitrogen cycle by metabolizing organic and inorganic nitrogen compounds. Denitrification is critical for maintenance of the global nitrogen cycle, through which nitrate (NO₃⁻) or nitrite (NO₂⁻) is reduced to gaseous nitrogen forms such as N₂ and nitrous oxide (N₂O) (19, 47). Decades of investigations into denitrifying bacteria have revealed their ecological impact (9), their molecular mechanisms of denitrification (13, 25, 47), and the industrial importance of removing nitrogenous contaminants from wastewater (31, 36). Bacterial denitrification is considered to comprise four successive reduction steps, each of which is catalyzed by NO₃⁻ reductase (Nar), NO₂⁻ reductase (Nir), nitric oxide (NO) reductase (Nor), and N₂O reductase (Nos). The reaction of each enzyme is linked to the electron transport chain on the cellular membrane and accompanies oxidative phosphorylation, implying that bacterial denitrification is of as much physiological significance as anaerobic respiration (25, 47). Most denitrifying bacteria are facultative anaerobes and respire with oxygen under aerobic conditions. Because denitrification is induced in the absence of oxygen, it is considered an alternative mechanism of energy conservation that has evolved as an adaptation to anaerobic circumstances (13, 47).Nitrite and NO are hazardous to bacteria, since they generate highly reactive nitrogen species (RNS) under physiological conditions and damage cellular DNA, lipid, and proteins (28, 37). Denitrifying bacteria are thought to be threatened by RNS since they reduce NO₃⁻ to generate NO₂⁻ and NO as denitrifying intermediates. Furthermore, denitrifying bacteria often inhabit environments where they are exposed to NO₂⁻ and NO and hence high levels of RNS. Recent reports suggest that pathogenic bacteria invading animal tissues are attacked by NO generated by macrophages (12). Such bacteria involve denitrifiers, and some of them, for example, Neisseria meningitidis (1) and Pseudomonas aeruginosa, acquire resistance to NO by producing Nor (44). The utilization (reduction) of NO by Brucella increases the survival of infected mice (2). These examples suggest that production of a denitrifying mechanism affects bacterial survival of threats from both endogenous and extracellular RNS. However, the mechanism of RNS tolerance induced by denitrifying bacteria is not fully understood.Ubiquitous gram-negative Ochrobactrum strains are widely distributed in soils and aqueous environments, where they biodegrade aromatic compounds (11), organophosphorus pesticides (45), and other hydrocarbons (38) and remove heavy metal ions such as chromium and cadmium (24). Having been isolated from clinical specimens, Ochrobactrum anthropi is currently recognized as an emerging opportunistic pathogen, although relatively little is known about its pathogenesis and factors contributing to its virulence (7, 30). Manipulation systems have been developed to investigate these issues at the molecular genetic level (33). Some O. anthropi strains have been identified as denitrifiers (21), although the denitrifying properties of these strains have not been investigated in detail. This study was undertaken to examine the denitrifying properties of O. anthropi in more detail. O. anthropi YD50.2 was selected for this study and was isolated herein. The strain denitrified high levels of NO₂⁻ (up to 40 mM) to dinitrogen under anaerobic conditions. The strain was highly resistant to acidified NO₂⁻ under nondenitrifying aerobic conditions. These results indicate that O. anthropi YD50.2 has mechanisms that produce tolerance to RNS.     

Proliferation Capacity and Cytotoxic Activity Are Mediated by Functionally and Phenotypically Distinct Virus-Specific CD8 T Cells Defined by Interleukin-7Rα (CD127) and Perforin Expression

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127⁺ perforin⁻/CD127⁻ perforin⁺, and CD127⁻/perforin⁻ CD8 T cells, respectively. CD127⁻/perforin⁻ and CD127⁻/perforin⁺ cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin⁺/IL-2⁻ or perforin⁻/IL-2⁺ cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127⁺/IL-2-secreting) and cytotoxic (perforin⁺) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.Cytotoxic CD8 T cells are a fundamental component of the immune response against viral infections and mediate an important role in immunosurveillance (7, 10, 55), and the induction of vigorous CD8 T-cell responses after vaccination is thought to be a key component of protective immunity (37, 41, 49, 50, 58, 60, 69). Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules containing perforin (pore-forming protein) and several granule-associated proteases, including granzymes (Grms) (5, 15, 20, 44). Several studies have recently advanced the characterization of the mechanism of granule-dependent cytotoxic activity and performed a comprehensive investigation of the content of cytotoxic granules in human virus-specific CD8 T cells (2, 19, 29, 44, 53).Heterogeneous profiles of cytotoxic granules have been identified in different virus-specific memory CD8 T cells and associated with distinct differentiation stages of memory CD8 T cells (2, 19, 29, 44). Furthermore, we have observed a hierarchy among the cytotoxic granules in setting the efficiency of cytotoxic activity and demonstrated that perforin (and to a lesser extent GrmB) but not GrmA or GrmK were associated with cytotoxic activity (29). Recently, a novel mechanism of perforin-dependent granule-independent CTL cytotoxicity has also been demonstrated (45).Major advances in the characterization of antigen (Ag)-specific CD4 and CD8 T cells have been made recently and have aimed at identifying functional profiles that may correlate with protective CD8 T-cell responses (1, 3, 4, 12, 13, 24, 28, 36-38, 40, 41, 49, 50, 56-58, 60, 64, 68). In particular, the functional characterization of antigen-specific T cells was mainly performed on the basis of (i) the pattern of cytokines secreted (i.e., gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-2 [IL-2], or macrophage inflammatory protein 1β [MIP-1β]), (ii) the proliferation capacity, and (iii) the cytotoxic capacity (13, 28, 59). Of note, degranulation activity (i.e., CD107a mobilization following specific stimulation) has been used as a surrogate marker of cytotoxic activity (11, 13).The term “polyfunctional” has been used to define T-cell immune responses that, in addition to typical effector functions such as secretion of IFN-γ, TNF-α, or MIP-1β and cytotoxic activity (measured by the degranulation capacity), comprise distinct T-cell populations able to secrete IL-2 and retain proliferation capacity (13, 28, 49, 50). Some evidence indicates that a hallmark of protective immune responses is the presence of polyfunctional T-cell responses (59). Furthermore, the ability to secrete IL-2 was shown to be linked to proliferation capacity, and both factors have been associated with protective antiviral immunity (13, 28, 49, 50). Although a lack of correlation between degranulation activity and GrmB expression was reported in mice (65), the relationship between degranulation activity and perforin expression has never been comprehensively investigated in mice and in humans.The private α chain of the IL-7 receptor (IL-7Rα, also called CD127) has been suggested to selectively identify CD8 T cells that will become long-lived memory cells (6, 34, 36). Moreover, it was shown in mice (34, 36) and humans (14, 48, 63) that the CD127^high memory-precursor CD8 T cells produced IL-2 in contrast to CD127^low effector CD8 T cells. Of interest, CD127 expression has also been shown to correlate with Ag-specific proliferation capacity in mice (34, 36). A similar correlation was observed in humans, although only for polyclonal stimulations (48). With the exception of studies performed in HIV-1 infection, where an association between CD127 expression and HIV-1 viremia has been shown (21, 22, 42, 48, 54), very limited information is available on the CD127 expression in human virus-specific CD8 T cells other that HIV-1.Although cytotoxic activity and proliferation capacity are key components of the antiviral cellular immune response, the relationship between these functions has been only investigated in nonprogressive HIV-1 infection (46), where these two functions were shown to be related. However, it still remains to be determined whether these functions are mediated by the same or by different T-cell populations.In the present study, we performed a comprehensive characterization of virus-specific CD8 T-cell responses against HIV-1, cytomegalovirus (CMV), Epstein Barr virus (EBV), and influenza virus (Flu) in order to (i) analyze the degree of concordance between degranulation activity and perforin/Grm expression; (ii) identify the relevance of CD127 in identifying virus-specific CD8 T cells endowed with proliferation capacity; (iii) delineate the relationship between proliferation capacity, cytotoxic activity, activation/differentiation stage, and level of exhaustion of CD8 T cells; and (iv) determine the influence of antigen exposure in shaping the functional composition of virus-specific CD8 T cells.Our data indicate that cytotoxic (as defined by perforin expression) and proliferating (as defined by CD127 expression or IL-2 secretion) virus-specific CD8 T cells are contained within distinct CD8 T-cell populations. Furthermore, the proportion of proliferating and cytotoxic T cells within a given virus-specific CD8 T-cell population appears to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferative capacity, differentiation stage, and Ag exposure of memory CD8 T cells.     

Pseudomonas Exotoxin A-Mediated Apoptosis Is Bak Dependent and Preceded by the Degradation of Mcl-1

Pseudomonas exotoxin A (PE) is a bacterial toxin that arrests protein synthesis and induces apoptosis. Here, we utilized mouse embryo fibroblasts (MEFs) deficient in Bak and Bax to determine the roles of these proteins in cell death induced by PE. PE induced a rapid and dose-dependent induction of apoptosis in wild-type (WT) and Bax knockout (Bax^−/−) MEFs but failed in Bak knockout (Bak^−/−) and Bax/Bak double-knockout (DKO) MEFs. Also a loss of mitochondrial membrane potential was observed in WT and Bax^−/− MEFs, but not in Bak^−/− or in DKO MEFs, indicating an effect of PE on mitochondrial permeability. PE-mediated inhibition of protein synthesis was identical in all 4 cell lines, indicating that differences in killing were due to steps after the ADP-ribosylation of EF2. Mcl-1, but not Bcl-x_L, was rapidly degraded after PE treatment, consistent with a role for Mcl-1 in the PE death pathway. Bak was associated with Mcl-1 and Bcl-x_L in MEFs and uncoupled from suppressed complexes after PE treatment. Overexpression of Mcl-1 and Bcl-x_L inhibited PE-induced MEF death. Our data suggest that Bak is the preferential mediator of PE-mediated apoptosis and that the rapid degradation of Mcl-1 unleashes Bak to activate apoptosis.Apoptosis is a mode of cell death utilized by multicellular organisms to remove unwanted cells. Also, many different cancer treatments, including chemotherapy and radiotherapy, induce apoptosis and result in the destruction of tumor cells. In some cases, apoptosis resistance can contribute to the failure of chemotherapy (14, 20, 24). Immunotoxins are a class of antitumor agents in which a powerful protein toxin is brought to the cancer cell by an antibody or an antibody fragment (for reviews, see references 28, 29, and 32). Several immunotoxins are currently in clinical trials, and one of these, BL22, targeting CD22, has shown excellent activity in drug-resistant hairy-cell leukemia (18, 19). Also, a fusion protein in which a fragment of diphtheria toxin is fused to the cytokine interleukin 2 (IL-2) (Ontak) is approved for the treatment of cutaneous T-cell lymphoma (26). Several studies carried out to determine how protein toxins and immunotoxins containing these toxins kill target cells have reported caspase activation (13, 16, 17, 30, 33). However, the steps leading up to caspase activation by these toxins that inhibit protein synthesis have not been elucidated.Bcl-2 family members are essential regulators of the mitochondrial (intrinsic) apoptosis pathway (1, 21). Proteins of this family have been divided into pro- and antiapoptotic proteins. Antiapoptotic proteins include the multi-Bcl-2 homology (BH) domain proteins Bcl-2, Bcl-x_L, Bcl-w, Mcl-1, Bcl-b, and Bcl2a1. Proapoptotic members can be further classified into two subfamilies, the multi-BH domain Bax homologues, including Bax, Bak, and Bok, and the BH3-only proteins, including Nbk/Bik, Noxa, Hrk, Bad, Bim, Puma, and Bmf. Bax and Bak are the most extensively studied central mediators in the mitochondrial apoptosis pathway (4, 6). Various stimuli, including pathogens, toxic drugs, irradiation, and starvation, induce a conformational change and activation of Bak/Bax, usually via BH3-only proapoptosis proteins. This results in the disruption of mitochondrial membranes and the release of apoptotic factors, such as cytochrome c, SMAC, and apoptosis-inducing factor, which lead to the activation of effector caspases (5, 37, 40, 42, 43).The roles of Bax and Bak can be redundant or nonredundant, depending on the apoptotic stimuli. Bak and Bax can compensate for each other in apoptosis induced by staurosporine, etoposide, UV irradiation, serum deprivation, tBid, Bim, Bad, or Noxa (37, 43). Bak plays an essential role for apoptosis induced by Semliki Forest virus, gliotoxin, Bcl-x_S, and vinblastine (22, 27, 34, 35), while Bax is favored for apoptosis induced by Nbk/Nik, a combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and ionizing irradiation, or TRAIL and 5-fluorouracil (5-FU) (9, 10, 36, 38). Silencing of either Bak or Bax resulted in resistance to apoptosis induced by Neisseria gonorrhoeae and cisplatin (15). Sometimes the same stimulus may result in different outcomes in different cell types. NBK/Bik mediated Bax-dependent cell death in one study (9), while in another study, NBK/Bik activated BAK-mediated apoptosis (31).In the current study, we utilized mutant mouse embryo fibroblasts (MEFs) deficient in Bak, Bax, or both proteins and provided evidence for an essential role of Bak in apoptosis induced by Pseudomonas exotoxin A (PE) and other protein synthesis inhibitors. We found that Bak^−/− cells are resistant to killing by PE and that Mcl-1, which binds to Bak, controls apoptosis induced by PE.     

Requirement of JIP1-Mediated c-Jun N-Terminal Kinase Activation for Obesity-Induced Insulin Resistance

The c-Jun NH₂-terminal kinase (JNK) interacting protein 1 (JIP1) has been proposed to act as a scaffold protein that mediates JNK activation. However, recent studies have implicated JIP1 in multiple biochemical processes. Physiological roles of JIP1 that are related to the JNK scaffold function of JIP1 are therefore unclear. To test the role of JIP1 in JNK activation, we created mice with a germ line point mutation in the Jip1 gene (Thr¹⁰³ replaced with Ala) that selectively blocks JIP1-mediated JNK activation. These mutant mice exhibit a severe defect in JNK activation caused by feeding of a high-fat diet. The loss of JIP1-mediated JNK activation protected the mutant mice against obesity-induced insulin resistance. We conclude that JIP1-mediated JNK activation plays a critical role in metabolic stress regulation of the JNK signaling pathway.Diet-induced obesity causes insulin resistance and metabolic syndrome, which can lead to β-cell dysfunction and type 2 diabetes (15). It is established that feeding mice a high-fat diet (HFD) causes activation of c-Jun NH₂-terminal kinase 1 (JNK1) (10). Moreover, Jnk1^−/− mice are protected against the effects of HFD-induced insulin resistance (10). Together, these observations indicate that JNK1 plays a critical role in the metabolic stress response. However, the mechanism that accounts for HFD-induced JNK1 activation is unclear. Recent studies have implicated the JIP1 scaffold protein in JNK1 activation caused by metabolic stress (23, 39).JIP1 can assemble a functional JNK activation module composed of a mitogen-activated protein kinase (MAPK) kinase kinase (a member of the mixed-lineage protein kinase [MLK] group), the MAPK kinase MKK7, and JNK (40, 42). This complex may be relevant to JNK activation caused by metabolic stress (23, 39). Indeed, MLK-deficient mice (14) and JIP1-deficient mice (13) exhibit defects in HFD-induced JNK activation and insulin resistance.The protection of Jip1^−/− mice against the effects of being fed an HFD may be mediated by loss of the JNK scaffold function of JIP1. However, JIP1 has also been reported to mediate other biochemical processes that would also be disrupted in Jip1^−/− mice. For example, JIP1 interacts with AKT and has been implicated in the mechanism of AKT activation (8, 17, 18, 34). Moreover, JIP1 interacts with members of the Src and Abl tyrosine kinase families (4, 16, 24), the lipid phosphatase SHIP2 (44), the MAPK phosphatase MKP7 (43), β-amyloid precursor protein (20, 31), the small GTPase regulatory proteins Ras-GRF1, p190-RhoGEF, RalGDS, and Tiam1 (2, 8, 21), ankyrin G (35), molecular chaperones (35), and the low-density-lipoprotein-related receptors LRP1, LRP2, and LRP8 (7, 37). JIP1 also interacts with other scaffold proteins, including the insulin receptor substrate proteins IRS1 and IRS2 (35). Finally, JIP1 may act as an adapter protein for kinesin-mediated (11, 12, 16, 38, 42) and dynein-mediated (35) trafficking on microtubules. The JNK scaffold properties of JIP1 therefore represent only one of the possible biochemical functions of JIP1 that are disrupted in Jip1^−/− mice.The purpose of this study was to test the role of JIP1 as a JNK scaffold protein in the response of mice to being fed an HFD. Our approach was to examine the effect of a point mutation that selectively prevents JIP1-induced JNK activation. It is established that phosphorylation of JIP1 on Thr¹⁰³ is required for JIP1-mediated JNK activation by the MLK pathway (25). Consequently, the phosphorylation-defective Thr103Ala JIP1 protein does not activate JNK (25). Here we describe the analysis of mice with a point mutation in the Jip1 gene that replaces the JIP1 phosphorylation site Thr¹⁰³ with Ala. We show that this mutation suppresses HFD-induced JNK activation and insulin resistance. These data demonstrate that JNK activation mediated by the JIP1 scaffold complex contributes to the response of mice to an HFD.     

Neuronal Protein Tyrosine Phosphatase 1B Deficiency Results in Inhibition of Hypothalamic AMPK and Isoform-Specific Activation of AMPK in Peripheral Tissues

PTP1B^−/− mice are resistant to diet-induced obesity due to leptin hypersensitivity and consequent increased energy expenditure. We aimed to determine the cellular mechanisms underlying this metabolic state. AMPK is an important mediator of leptin''s metabolic effects. We find that α1 and α2 AMPK activity are elevated and acetyl-coenzyme A carboxylase activity is decreased in the muscle and brown adipose tissue (BAT) of PTP1B^−/− mice. The effects of PTP1B deficiency on α2, but not α1, AMPK activity in BAT and muscle are neuronally mediated, as they are present in neuron- but not muscle-specific PTP1B^−/− mice. In addition, AMPK activity is decreased in the hypothalamic nuclei of neuronal and whole-body PTP1B^−/− mice, accompanied by alterations in neuropeptide expression that are indicative of enhanced leptin sensitivity. Furthermore, AMPK target genes regulating mitochondrial biogenesis, fatty acid oxidation, and energy expenditure are induced with PTP1B inhibition, resulting in increased mitochondrial content in BAT and conversion to a more oxidative muscle fiber type. Thus, neuronal PTP1B inhibition results in decreased hypothalamic AMPK activity, isoform-specific AMPK activation in peripheral tissues, and downstream gene expression changes that promote leanness and increased energy expenditure. Therefore, the mechanism by which PTP1B regulates adiposity and leptin sensitivity likely involves the coordinated regulation of AMPK in hypothalamus and peripheral tissues.Protein tyrosine phosphatase 1B (PTP1B) belongs to a family of tyrosine phosphatases with diverse roles in eukaryotes (2, 4). PTP1B attenuates insulin signaling by dephosphorylating the insulin receptor (19, 22, 61) and possibly IRS-1 (9, 23) and leptin signaling by dephosphorylating JAK2, which phosphorylates the leptin receptor and associated substrates (10, 45, 67). PTP1B-deficient mice are insulin hypersensitive, lean, and resistant to diet-induced obesity (20, 36) due, at least in part, to increased energy expenditure (36). The leanness can be explained by the absence of PTP1B in neurons, because neuron-specific PTP1B^−/− mice also have reduced body weight and adiposity and increased energy expenditure (6). In contrast, muscle- and liver-specific PTP1B-deficient mice have normal body weight with improved insulin sensitivity, whereas adipose-PTP1B-deficient mice have increased body weight (6, 15, 16). These data suggest that PTP1B in peripheral tissues such as muscle and liver is an important mediator of peripheral insulin sensitivity, whereas PTP1B in the nervous system plays a critical role in regulating energy expenditure and adiposity (6).The adipocyte-derived hormone leptin plays an essential role in regulating energy homeostasis by acting on multiple tissues, most importantly the hypothalamus, to regulate food intake and energy expenditure (1). PTP1B^−/− mice have enhanced basal and leptin-stimulated hypothalamic STAT3 phosphorylation and are hypersensitive to leptin''s effect on food intake and body weight (10, 67). The overexpression of PTP1B in heterologous cells dose dependently reduces the leptin-induced phosphorylation of JAK2 and STAT3 and inhibits leptin-stimulated STAT3-dependent reporter gene activation (10, 35, 39, 67). These and other data established that enhanced leptin sensitivity contributes to the leanness in PTP1B^−/− mice. We sought to determine the cellular mechanisms underlying the altered energy homeostasis in the setting of PTP1B deficiency.AMP-activated protein kinase (AMPK) is a major mediator of leptin''s metabolic effects (43, 44). AMPK is a fuel-sensing enzyme complex activated by cellular stresses that increase AMP or deplete ATP, including hypoxia, ischemia, glucose deprivation, uncouplers of oxidative phosphorylation, exercise, and muscle contraction (66). AMPK also is activated by the antidiabetic drugs metformin (68) and the thiazolidinediones (21). Mechanisms involved in AMPK activation include (i) the binding of AMP to an allosteric site on the γ subunit, which renders the holoenzyme resistant to inactivating serine phosphatases and also may have direct allosteric effects on kinase activity (55), and (ii) phosphorylation by upstream AMPK kinases of the α (catalytic) subunits on Thr172, which is essential for kinase activity (29). Once activated, AMPK phosphorylates multiple downstream substrates, leading to the inhibition of ATP-utilizing pathways, such as fatty acid synthesis, and the activation of ATP-generating pathways, including fatty acid oxidation (34).The phosphorylation of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC) by AMPK results in the inhibition of ACC activity, decreased malonyl-CoA content, and a subsequent increase in fatty acid oxidation in skeletal muscle caused by the disinhibition of carnitine palmitoyltransferase 1 (27, 52, 62). The leptin stimulation of muscle fatty acid oxidation is mediated by AMPK (44). AMPK also is an important regulator of muscle mitochondrial biogenesis and function (7, 37, 48, 58, 63). This may, in part, be mediated by peroxisome proliferator-activated receptor γ (PPARγ)-coactivator 1α (PGC-1α), because AMPK induces the expression and phosphorylation of PGC-1α, which regulates mitochondrial biogenesis and muscle fiber type (31).In addition to a role for AMPK in leptin action in peripheral tissues, the inhibition of hypothalamic AMPK activity by leptin plays an important role in mediating leptin''s effect on food intake and energy homeostasis (43). This appears to involve neurons that express neuropeptide Y (NPY) and agouti-related peptide (AgRP), since the expression of constitutively active AMPK in the basomedial hypothalamus augments NPY/AgRP expression (43). Furthermore, the deletion of the AMPK α2 catalytic subunit specifically in these neurons results in leanness, whereas deletion in proopiomelanocortin (POMC)-expressing neurons results in mild obesity (13).To determine whether alterations in AMPK contribute to increased energy expenditure and leanness in PTP1B^−/− mice, we investigated the AMPK pathway in peripheral tissues and hypothalamus. We demonstrate that the global absence of PTP1B alters AMPK and downstream biological processes in multiple tissues, and that neuronal PTP1B regulates AMPK activity in peripheral tissues in an isoform-specific manner. Our data establish a novel link between PTP1B and AMPK, two signaling molecules that are critical in the regulation of energy homeostasis.     

Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

We previously reported that CD4C/human immunodeficiency virus (HIV)^Nef transgenic (Tg) mice, expressing Nef in CD4⁺ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4⁺ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4⁺ T cells, DCs, and macrophages (CD4E/HIV^Nef); in CD4⁺ T cells and DCs (mCD4/HIV^Nef and CD4F/HIV^Nef); in macrophages and DCs (CD68/HIV^Nef); or mainly in DCs (CD11c/HIV^Nef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV^Nef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4⁺ T cells showed CD4⁺ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4⁺ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.The major cell targets and reservoirs for human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection in vivo are CD4⁺ T lymphocytes and antigen-presenting cells (macrophages and dendritic cells [DC]) (21, 24, 51). The cell specificity of these viruses is largely dependent on the expression of CD4 and of its coreceptors, CCR5 and CXCR-4, at the cell surface (29, 66). Infection of these immune cells leads to the severe disease, AIDS, showing widespread manifestations, including progressive immunodeficiency, immune activation, CD4⁺ T-cell depletion, wasting, dementia, nephropathy, heart and lung diseases, and susceptibility to opportunistic pathogens, such as Candida albicans (1, 27, 31, 37, 41, 82, 93, 109). It is reasonable to assume that the various pathological changes in AIDS result from the expression of one or many HIV-1/SIV proteins in these immune target cells. However, assigning the contribution of each infected cell subset to each phenotype has been remarkably difficult, despite evidence that AIDS T-cell phenotypes can present very differently depending on the strains of infecting HIV-1 or SIV or on the cells targeted by the virus (4, 39, 49, 52, 72). For example, the T-cell-tropic X4 HIV strains have long been associated with late events and severe CD4⁺ T-cell depletion (22, 85, 96). However, there are a number of target cell subsets expressing CD4 and CXCR-4, and identifying which one is responsible for this enhanced virulence has not been achieved in vivo. Similarly, the replication of SIV in specific regions of the thymus (cortical versus medullary areas), has been associated with very different outcomes but, unfortunately, the critical target cells of the viruses were not identified either in these studies (60, 80). The task is even more complex, because HIV-1 or SIV can infect several cell subsets within a single cell population. In the thymus, double (CD4⁻ CD8⁻)-negative (DN) or triple (CD3⁻ CD4⁻ CD8⁻)-negative (TN) T cells, as well as double-positive (CD4⁺ CD8⁺) (DP) T cells, are infectible by HIV-1 in vitro (9, 28, 74, 84, 98, 99, 110) and in SCID-hu mice (2, 5, 91, 94). In peripheral organs, gut memory CCR5⁺ CD4⁺ T cells are primarily infected with R5 SIV, SHIV, or HIV, while circulating CD4⁺ T cells can be infected by X4 viruses (13, 42, 49, 69, 70, 100, 101, 104). Moreover, some detrimental effects on CD4⁺ T cells have been postulated to originate from HIV-1/SIV gene expression in bystander cells, such as macrophages or DC, suggesting that other infected target cells may contribute to the loss of CD4⁺ T cells (6, 7, 32, 36, 64, 90).Similarly, the infected cell population(s) required and sufficient to induce the organ diseases associated with HIV-1/SIV expression (brain, heart, and kidney) have not yet all been identified. For lung or kidney disease, HIV-specific cytotoxic CD8⁺ T cells (1, 75) or infected podocytes (50, 95), respectively, have been implicated. Activated macrophages have been postulated to play an important role in heart disease (108) and in AIDS dementia (35), although other target cells could be infected by macrophage-tropic viruses and may contribute significantly to the decrease of central nervous system functions (11, 86, 97), as previously pointed out (25).Therefore, because of the widespread nature of HIV-1 infection and the difficulty in extrapolating tropism of HIV-1/SIV in vitro to their cell targeting in vivo (8, 10, 71), alternative approaches are needed to establish the contribution of individual infected cell populations to the multiorgan phenotypes observed in AIDS. To this end, we developed a transgenic (Tg) mouse model of AIDS using a nonreplicating HIV-1 genome expressed through the regulatory sequences of the human CD4 gene (CD4C), in the same murine cells as those targeted by HIV-1 in humans, namely, in immature and mature CD4⁺ T cells, as well as in cells of the macrophage/DC lineages (47, 48, 77; unpublished data). These CD4C/HIV Tg mice develop a multitude of pathologies closely mimicking those of AIDS patients. These include a gradual destruction of the immune system, characterized among other things by thymic and lymphoid organ atrophy, depletion of mature and immature CD4⁺ T lymphocytes, activation of CD4⁺ and CD8⁺ T cells, susceptibility to mucosal candidiasis, HIV-associated nephropathy, and pulmonary and cardiac complications (26, 43, 44, 57, 76, 77, 79, 106). We demonstrated that Nef is the major determinant of the HIV-1 pathogenicity in CD4C/HIV Tg mice (44). The similarities of the AIDS-like phenotypes of these Tg mice to those in human AIDS strongly suggest that such a Tg mouse approach can be used to investigate the contribution of distinct HIV-1-expressing cell populations to their development.In the present study, we constructed and characterized five additional mouse Tg strains expressing Nef, through distinct regulatory elements, in cell populations more restricted than in CD4C/HIV Tg mice. The aim of this effort was to assess whether, and to what extent, the targeting of Nef in distinct immune cell populations affects disease development and progression.     

Induction of Robust Cellular and Humoral Virus-Specific Adaptive Immune Responses in Human Immunodeficiency Virus-Infected Humanized BLT Mice

The generation of humanized BLT mice by the cotransplantation of human fetal thymus and liver tissues and CD34⁺ fetal liver cells into nonobese diabetic/severe combined immunodeficiency mice allows for the long-term reconstitution of a functional human immune system, with human T cells, B cells, dendritic cells, and monocytes/macrophages repopulating mouse tissues. Here, we show that humanized BLT mice sustained high-level disseminated human immunodeficiency virus (HIV) infection, resulting in CD4⁺ T-cell depletion and generalized immune activation. Following infection, HIV-specific humoral responses were present in all mice by 3 months, and HIV-specific CD4⁺ and CD8⁺ T-cell responses were detected in the majority of mice tested after 9 weeks of infection. Despite robust HIV-specific responses, however, viral loads remained elevated in infected BLT mice, raising the possibility that these responses are dysfunctional. The increased T-cell expression of the negative costimulator PD-1 recently has been postulated to contribute to T-cell dysfunction in chronic HIV infection. As seen in human infection, both CD4⁺ and CD8⁺ T cells demonstrated increased PD-1 expression in HIV-infected BLT mice, and PD-1 levels in these cells correlated positively with viral load and inversely with CD4⁺ cell levels. The ability of humanized BLT mice to generate both cellular and humoral immune responses to HIV will allow the further investigation of human HIV-specific immune responses in vivo and suggests that these mice are able to provide a platform to assess candidate HIV vaccines and other immunotherapeutic strategies.An ideal animal model of human immunodeficiency virus (HIV) infection remains elusive. Nonhuman primates that are susceptible to HIV infection typically do not develop immunodeficiency (63), and although the simian immunodeficiency virus (SIV) infection of rhesus macaques has provided many critically important insights into retroviral pathogenesis (30), biological and financial considerations have created some limitations to the wide dissemination of this model. The great need for an improved animal model of HIV itself recently has been underscored by the disappointing results of human trials of MRKAd5, an adenovirus-based HIV type 1 (HIV-1) vaccine. This vaccine was not effective and actually may have increased some subjects'' risk of acquiring HIV (53). In the wake of these disappointing results, there has been increased interest in humanized mouse models of HIV infection (54). The ability of humanized mouse models to test candidate vaccines or other immunomodulatory strategies will depend critically on the ability of these mice to generate robust anti-HIV human immune responses.Mice have provided important model systems for the study of many human diseases, but they are unable to support productive HIV infection, even when made to express human coreceptors for the virus (7, 37, 52). A more successful strategy to humanize mice has been to engraft human immune cells and/or tissues into immunodeficient severe combined immunodeficiency (SCID) or nonobese diabetic (NOD)/SCID mice that are unable to reject xenogeneic grafts (39, 42, 57). Early versions of humanized mice supported productive HIV infection and allowed investigators to begin to address important questions in HIV biology in vivo (23, 40, 43-45). More recently, human cord blood or fetal liver CD34⁺ cells have been used to reconstitute Rag2^−/− interleukin-2 receptor γ chain-deficient (γ_c^−/−) and NOD/SCID/γ_c^−/− mice, resulting in higher levels of sustained human immune cell engraftment (27, 29, 61). These mice have allowed for stable, disseminated HIV infection (2, 4, 24, 65, 67), including mucosal transmission via vaginal and rectal routes (3). These mice recently have been used to demonstrate an important role for Treg cells in acute HIV infection (29) and to demonstrate that the T-cell-specific delivery of antiviral small interfering RNA is able to suppress HIV replication in vivo (31). These mice also have demonstrated some evidence of adaptive human immune responses, including the generation of HIV-specific antibody responses in some infected mice (2, 65), and some evidence of humoral and cell-mediated responses to non-HIV antigens or pathogens (24, 61). Most impressively, Rag2^−/− γ_c^−/− mice reconstituted with human fetal liver-derived CD34⁺ cells have generated humoral responses to dengue virus infection that demonstrated both class switching and neutralizing capacity (32). In spite of these advances, however, these models have not yet been reported to generate de novo HIV-specific cell-mediated immune responses, which are considered to be a crucial arm of host defense against HIV infection in humans.In contrast to humanized mouse models in which only human hematopoietic cells are transferred into immunodeficient mice, the surgical implantation of human fetal thymic and liver tissue has been performed in addition to the transfer of human hematopoietic stem cells (HSC) to generate mice in which human T cells are educated by autologous human thymic tissue rather than by the xenogeneic mouse thymus. Melkus and colleagues refer to mice they have reconstituted in this way as NOD/SCID-hu BLT (for bone marrow, liver, and thymus), or simply BLT, mice (41). We previously referred to mice that we have humanized in a similar way as NOD/SCID mice cotransplanted with human fetal thymic and liver tissues (Thy/Liv) and CD34⁺ fetal liver cells (FLC) (33, 60) but now adopt the designation BLT mice as well. BLT mice demonstrate the robust repopulation of mouse lymphoid tissues with functional human T lymphocytes (33, 41, 60) and can support the rectal and vaginal transmission of HIV (13, 59). Further, BLT mice demonstrate antigen-specific human immune responses against non-HIV antigens and/or pathogens (41, 60). The ability of these mice to generate human immune responses against HIV, however, has not yet been reported. In this study, we investigated whether the provision of autologous human thymic tissue in BLT mice generated by the cotransplantion of human fetal Thy/Liv tissues and CD34⁺ FLC would allow for the maturation of human T cells in humanized mice capable of providing improved cellular responses to HIV as well as providing adequate help for improved humoral responses. To describe the cells contributing to human immune responses in BLT mice, we also characterized the phenotypes of multiple subsets of T cells, B cells, dendritic cells (DCs), and monocytes/macrophages present in uninfected humanized mice. The generation of robust HIV-directed human cellular and humoral immune responses in these mice would further demonstrate the ability of humanized mice to provide a much needed platform for the evaluation of HIV vaccines and other novel immunomodulatory strategies.     

Evolution of the HIV-1 env Gene in the Rag2?/? γC?/? Humanized Mouse Model

Rag2^−/− γ_C^−/− mice transplanted with human hematopoietic stem cells (DKO-hu-HSC mice) mimic aspects of human infection with human immunodeficiency virus type 1 (HIV-1), including sustained viral replication and CD4⁺ T-cell decline. However, the extent of HIV-1 evolution during long-term infection in these humanized mice, a key feature of the natural infection, has not been assessed fully. In this study, we examined the types of genotypic and phenotypic changes in the viral env gene that occur in the viral populations of DKO-hu-HSC mice infected with the CCR5-tropic isolate HIV-1_JRCSF for up to 44 weeks. The mean rate of divergence of viral populations in mice was similar to that observed in a cohort of humans during a similar period of infection. Many amino acid substitutions were common across mice, including losses of N-linked glycosylation sites and substitutions in the CD4 binding site and in CD4-induced epitopes, indicating common selective pressures between mice. In addition, env variants evolved sensitivity to antibodies directed at V3, suggesting a more open conformation for Env. This phenotypic change was associated with increased CD4 binding efficiency and was attributed to specific amino acid substitutions. In one mouse, env variants emerged that exhibited a CXCR4-tropic phenotype. These sequences were compartmentalized in the mesenteric lymph node. In summary, viral populations in these mice exhibited dynamic behavior that included sequence evolution, compartmentalization, and the appearance of distinct phenotypic changes. Thus, humanized mice offer a useful model for studying evolutionary processes of HIV-1 in a complex host environment.Animal models of HIV-1 infection are important tools for studying transmission, replication, and pathogenesis, as well as therapeutic intervention, of HIV-1 infection. Nonhuman primates such as rhesus macaques, infected with simian or chimeric simian/human immunodeficiency viruses (SIV or SHIV, respectively), represent well-characterized and highly relevant models; however, key limitations include expense, genetic variability of the host animals, and the fact that SIV, while closely related, is distinct from HIV-1. Therefore, small animal models that support HIV-1 infection and recapitulate many aspects of the human infection have been sought using several approaches.Recent approaches have involved the use of genetically immunodeficient mice that have been reconstituted using human-derived hematopoietic stem cells (HSC) (known as humanized mice). Several models have been developed based on this approach, including Rag2^−/− γ_C^−/− (DKO) and NOD/SCID/γ_C^−/− (NOG or NSG) mice transplanted with human HSC (DKO-hu-HSC or NOG-hu-HSC mice) (40, 92) and the NOD/SCID mouse with transplanted human fetal thymus and liver tissue in addition to HSC (62). These models all support HIV-1 infection (1, 3, 6, 30, 87, 96, 102; for a review of these models, see the work of Denton and Garcia [22]). The DKO-hu-HSC mouse lacks both recombination activating gene 2 (Rag2) and the cytokine receptor common gamma chain (γ_C), and as a result, it does not generate murine T, B, and natural killer (NK) cells but supports engraftment of HSC and differentiation of human myeloid and lymphoid lineages. Immune reconstitution in this model likely involves education of human T cells in the mouse thymus and dissemination of differentiated human lymphoid subsets into the peripheral blood and to multiple lymphoid tissues, including lymph nodes, spleen, and bone marrow (92). The DKO-hu-HSC mouse, along with the other humanized mouse models, has been used in studies of transmission (5, 21), pathogenesis (43), and viral inhibition (16, 21, 53, 88, 94).One important feature of HIV-1 infection is the diversification and evolution of the viral genome over the course of infection. Diversification occurs most prominently in the envelope (env) gene, which encodes the viral surface glycoprotein (Env). Env mediates viral entry into cells through attachment to the primary receptor CD4, which primes Env for engagement with a coreceptor, either CCR5 or CXCR4, triggering virion fusion with the cellular plasma membrane (54). HIV-1 infection is typically established by one or a few CCR5-tropic (R5) variants that give rise to an initially homogenous viral population, which then diversifies over the course of chronic infection (45, 84). Diversification of Env results from immune selective pressures (27), isolation in or adaptation to different cellular and anatomical compartments (20, 28, 33, 46, 51), and selection for altered CD4 affinity (72, 90, 95) and coreceptor tropism (26, 39). In many cases, during late-stage infection, variants emerge from the R5 virus population that are CXCR4 tropic (X4), an event that is often associated with accelerated CD4 T-cell loss and progression to AIDS (9, 18, 89). In an effort to determine if any of these aspects of HIV-1 evolution are exhibited in the humanized mouse model, we examined the extent of HIV-1 diversification and the types of evolutionary changes that occur in env in mice infected with CCR5-tropic HIV-1 for up to 44 weeks.Sampling of viral env variants from the peripheral blood plasma over the course of the infection revealed increasing diversity and divergence of the viral population at rates similar to those observed in natural infection. Mutations were identified that affected Env conformation and sensitivity to neutralizing antibodies, CXCR4 coreceptor use, and potential N-linked glycosylation sites. Other mutations potentially affecting the Env phenotype were identified in CD4 binding sites and CD4-induced epitopes. The patterns of substitutions indicated that certain sites were under selection, particularly in cases where the same substitution was identified in multiple mice.This study demonstrates the potential for studying HIV-1 evolution in the DKO-hu-HSC mouse model and also gives insight into the types of selective pressures driving HIV-1 env evolution in this host environment. These findings, while highlighting some of the limitations of this model, will help to inform its appropriate use for studying different aspects of HIV-1 infection, such as the evolutionary constraints placed on HIV-1 during natural infection and in the face of pharmacological and immunological inhibition.